Ashwagandha and Shatavari Extracts Dose-Dependently Reduce Menopause Symptoms, Vascular Dysfunction, and Bone Resorption in Postmenopausal Women: A Randomized, Double-Blind, Placebo-Controlled Study

Abstract

Objectives

In this study, we investigate the effects of dietary supplementation with standardized aqueous extracts of shatavari (Asparagus racemosus, Ar), ashwagandha (Withania somnifera, Ws), or their combination on menopausal symptoms, vascular dysfunction, bone turnover, and serum concentrations of inflammatory and oxidative stress markers in postmenopausal women.

Methods

Postmenopausal women aged 40–55 were enrolled in a double-blind randomized study to receive one of six treatments: placebo, Ar 250 mg/500 mg, Ws 250 mg/500 mg, or 500 mg extract combining Ar 250 mg and Ws 250 mg. Primary outcomes were changes in the menopause-specific quality of life (MENQOL) questionnaire, bone mineral density/bone turnover markers (BTMs), and reflection index (RI) after 24 weeks. Secondary outcomes included changes in serum inflammatory and oxidative stress markers, and evaluation of supplement safety and tolerability.

Results

Supplemented groups showed significant dose-dependent decrease MENQOL and RI compared with placebo (P < 0.0001). Women supplemented with Ws or Ar extracts had significantly decreased levels of the BTMs C-terminal telopeptide of type I collagen, bone alkaline phosphatase, and receptor activator of nuclear factor kappa-B ligand, and increased osteoprotegerin levels (P < 0.0001). Significantly decreased levels of inflammatory and oxidative stress markers high-sensitivity C-reactive protein and malondialdehyde, and increased glutathione and nitric oxide levels (P < 0.0001) were also observed.

Conclusions

Daily supplementation with Ws or Ar extracts dose-dependently reduces menopausal symptoms, vascular dysfunction, bone turnover/resorption, and estrogen deficiency-related inflammation and oxidative stress in postmenopausal women.

INTRODUCTION

Menopause is a natural phase for most women, characterized by the cessation of ovarian function and menstruation. However, it is frequently associated with symptoms that can adversely impact health and quality of life. It is estimated that the population of menopausal and postmenopausal women will increase to 1.2 billion by the year 2030, growing by 47 million added annually [1]. It is estimated that up to 85% of postmenopausal women have suffered at least one menopause-related symptom during their lifetime [2]. Vasomotor symptoms (e.g., hot flashes, night sweats), related to vascular endothelial dysfunction, are the primary symptoms of menopause experienced by a majority of women during the menopause transition [3].

Oestrogen deficiency in postmenopausal women not only leads to physiologic dysfunctions such as hot flashes, night sweats, palpitations, sleep disruption, fatigue, head and body aches, weight gain, vaginal dryness (linked to painful sex and diminished sexual satisfaction) and atrophy, but also psychological symptoms such as anxiety, depression, forgetfulness, irritability, and inability to concentrate [4,5,6]. Thus, menopause symptoms have a substantial effect on women’s quality of life and workplace performance. Additionally, bone mass and density decline with advancing age and the rate of decline is particularly high in postmenopausal women, thereby increasing risk for osteoporosis and bone fracture.

Aging is linked to chronic, low-grade systemic inflammation and oxidative stress, while the beneficial effects of oestrogen on bone and vascular endothelial function are partially due to its ability to reduce both inflammation and oxidative stress [7,8,9,10]. Production of proinflammatory cytokines that promote bone resorption, such as tumour necrosis factor-α (TNF-α) and receptor activator of nuclear factor kappa-B ligand (RANKL), are suppressed by oestrogens, while production of cytokines that discourage bone resorption, such as transforming growth factor-β (TGF-β) and osteoprotegerin (OPG), are enhanced by oestrogen [7,11]. Oxidative stress also favours bone loss as reactive oxygen species (ROS) reduce osteoblast (bone forming cells) differentiation and activity and promote bone resorption [12,13,14]. Oestrogen deficiency is associated with diminished glutathione (GSH) levels, the primary endogenous cellular antioxidant, and antioxidant defences, which contributes to increased oxidative stress and proinflammatory signalling [13] TNF-α and vascular inflammation are critical contributors to vascular endothelial dysfunction in aging and oestrogen deficiency, partly by decreasing the production and signalling of vascular endothelial nitric oxide (NO) [15]. In ovariectomized animals, vascular endothelial dysfunction is associated with increased ROS and reduced NO production, whereas treatment with oestrogen reduced ROS and vascular endothelial dysfunction [16]. In a population of healthy menopausal women, total antioxidant activity in blood of those experiencing hot flashes was lower than in those without this symptom [17], suggesting oxidative stress contributes to bothersome menopause symptoms.

Hormone replacement therapy (HRT) is a medical option available to address multiple adverse menopause symptoms and attenuate accelerated bone loss in postmenopausal women; however, HRT has been associated with increased risk for cardiovascular diseases and breast cancer [18], so many women seek complementary and alternative therapeutic options including plant-based therapies such as soy isoflavones and other sources of phytoestrogens. Phytoestrogens are plant-derived compounds that share structural and/or functional similarities with mammalian oestrogens and display varying degrees of estrogenic activity in the body [19], thus are anticipated to have pro-estrogenic activity in the face of oestrogen deficiency.

Withania somnifera (Ws), commonly known as ashwagandha, is considered the main rejuvenated tonic for males [20]. Ws exhibits broad therapeutic potential and is commonly used to reduce mental and physical dysfunctions associated with excess stress and anxiety [21,22,23]. In addition to its psychological benefits, research has shown that dietary supplementation with ashwagandha reduces inflammation, oxidative stress, and related symptoms, such as vascular dysfunction, while improving GSH levels in populations with elevated oxidative stress and inflammation [22,23,24,25].

Asparagus racemosus (Ar), commonly known as shatavari, is the primary herb recommended for female health in Ayurveda [26], the holistic healing system originating in India thousands of years ago still practiced today. Ar possesses a variety of phytochemical constituents such as steroidal sapogenins and saponins, including sarsasapogenin and shatavarins (sarsasapogenin glycosides), respectively, flavonoids, including isoflavones, as well as sterols, alkaloids, vitamins, and minerals [27,28]. The roots of Ar are commonly used therapeutically due in part to its phytoestrogenic as well as anti-inflammatory and antioxidant properties [26]. However, clinical evidence to support its therapeutic efficacy is lacking. This experience-based therapies require clinical investigation to guide evidence-based therapeutic uses.

In this study, we aimed to investigate dietary supplementation with standardized aqueous extracts of Ar and Ws, or their combination, on reducing menopause symptoms, vascular dysfunction, bone turnover, and serum concentrations of inflammatory and oxidative stress markers among postmenopausal women. The dosing was selected based on prior clinical research demonstrating reduced chronic stress and associated symptoms, inflammation, oxidative stress, and vascular dysfunction with daily supplementation of the Ws extract [22,24].

MATERIALS AND METHODS

Recruitment

This 24-week prospective, randomized, double-blind, placebo-controlled, parallel-group intervention study was conducted at Nizam’s Institute of Medical Sciences (NIMS), Hyderabad, India. The study received approved by the NIMS Institutional Ethics Committee on 02/04/2019 and took place from July 2019 to December 2020 (review letter no: EC/NIMS/2334/2019 38th ESGS No: 824/2018). The study was conducted in accordance with the Declaration of Helsinki (2013) and Guidelines for Clinical Trials on Pharmaceutical Products in India – GCP Guidelines issued by the Central Drugs Standard Control Organization, Ministry of Health, and Government of India. This study was registered with Clinical Trials Registry – India (CTRI) with the registration number: CTRI/2019/07/020347 (registered on 24/07/2019 – trial registered prospectively).

Inclusion criteria required participants to be menopausal (defined as no menstruation for 12 consecutive months), aged 40 to 55 years, with normal thyroid, liver, and kidney function, serum 25-hydroxy vitamin D levels above 20 ng/mL, no use of bisphosphonates in the past year, and a willingness to participate in the 24-week study. Exclusion criteria included individuals with a history or evidence of malignancy, surgical menopause, diagnosed mental illness, hypertension, diabetes mellitus, rheumatoid arthritis, coronary artery disease, hepatic or renal impairment, a history of metabolic bone disease or recent fractures, use of medications (calcitonin, raloxifene, or systemic glucocorticoids) within 3 months prior to the study, hormone or hormone-like replacement therapy within 6 months, or a glycated haemoglobin level above 8% in the past 3 months.

Study protocol

After screening, eligible participants were randomly assigned to one of six groups using computer-generated block randomization: 1) placebo, 2) Ws 250 mg, 3) Ws 500 mg, 4) Ar 250 mg, 5) Ar 500 mg, and 6) Ws 250 mg + Ar 250 mg. Written informed consent was obtained from all participants prior to their involvement in the study. The first investigational product, Sensoril^® (Kerry Group), is an aqueous extract derived from Ws roots and aerial parts, consisting of fifty small molecules, including 26 withanolides. Ten major secondary metabolites (9 withanolides and one flavonoid glycoside) are selected for quantification by ultra performance liquid chromatography with photodiode array detection and were in the range of 0.4%–1.0%. The second investigational product is an aqueous extract derived from Ar roots standardized to > 30% saponins.

Identical white opaque capsules, each containing either a placebo (microcrystalline cellulose), 125 mg or 250 mg of Ws extract with microcrystalline cellulose (as an excipient), or 125 mg or 250 mg of Ar extract with microcrystalline cellulose (as an excipient), were manufactured according to current good manufacturing practices and supplied by Natreon Inc. Capsules were dispensed by the research pharmacist in sequentially numbered containers based on a randomly allocated sequence. Participants were instructed to take two capsules daily, once in the morning and once in the evening after meals, for a period of 24 weeks. Participants reported to the study site at 8 a.m. after an overnight fast (8–10 hours) at weeks 0, 4, 8, 12, and 24. Blood samples were collected in 8.5 mL gold top BD Vacutainer serum separation tubes, mixed with a clotting activation agent by inverting the tubes at least five times, allowed to clot for 30 minutes (with tubes standing upright), and then centrifuged at 2,500 to 3,000 rpm for 15 minutes. The serum was then aliquoted, snap-frozen in liquid nitrogen, and stored at –80°C for future analysis. Participants were asked to report any adverse effects, which were documented in the case report form. Compliance with the supplementation regimen was assessed using the pill count method.

Primary outcomes

Menopause-specific quality of life

Women completed the validated menopause-specific quality of life (MENQOL) questionnaire [29] at weeks 0, 4, 8, 12, and 24. The MENQOL questionnaire comprises 29 items (symptoms) that participants report if they have experienced them over the past month and rated using a 7-point Likert scale ranging from 0 = “not at all bothered” to 6 = “extremely bothered.” Items are grouped into four domains: (1) vasomotor (3 items), (2) psychosocial (7 items), (3) physical (16 items), and (4) sexual (3 items). Scores in each domain were converted into an analysis score ranging from 1 (no symptoms) to 8 (extremely bothered) and evaluated individually. The mean MENQOL score was calculated as the average of domain means.

Vascular endothelial dysfunction (reflection index)

A salbutamol challenge test, using digital volume plethysmography, was conducted to assess vascular endothelial function at weeks 0, 4, 8, 12, and 24, following the methods described by Chowienczyk et al. [30] and Naidu et al. [31] at weeks 0, 4, 8, 12, and 24. Patients were examined in a supine position after 5 minutes of rest. A digital volume pulse (DVP) was obtained using a photoplethysmograph (Pulse Trace PCA2, PT200; Micro Medical) transmitting infrared light at 940 nm, placed on the index finger of the right hand. The signal from the plethysmograph was digitized using a 12-bit analogue-to-digital converter with a sampling frequency of 100 Hz. DVP waveforms were recorded over a 20-second period, and the height of the late systolic/early diastolic portion of the DVP was expressed as a percentage of the amplitude of the DVP to yield the reflection index (RI), following the procedure detailed by Millasseau et al. [32]. Three DVP recordings were taken, and the average RI value was computed. Following this, patients inhaled 400 mcg of salbutamol. After 15 minutes, three additional RI measurements were recorded, and the change in mean RI before and after salbutamol administration was used to evaluate endothelial function. A ≤ 6% change in RI post-salbutamol was considered indicative of endothelial dysfunction. The percentage change in RI in response to salbutamol is comparable to brachial artery flow-mediated dilation and effectively identifies subjects with vascular endothelial dysfunction [33].

Bone mineral density by dual energy X-ray absorptiometry

Bone mineral density (BMD) of the L1–L4 lumbar region of the spine and femoral neck was determined using a Lunar DPX dual-energy X-ray absorptiometer system (analysis version 11.40, GE Healthcare) at the basal and final visit (24 weeks). The readers remained blinded throughout the entire study period.

Systemic biomarkers of bone turnover markers

Bone turnover markers (BTMs) are useful for evaluating short-term (< 1 year) responses to interventions aimed at preventing bone loss, as changes in BMD typically occur gradually. Elevated BTM levels indicate increased bone turnover, which is associated with postmenopausal bone loss [34,35,36]. BTM included C-terminal telopeptide of type I collagen (CTX-1), bone alkaline phosphatase (BALP), RANKL, OPG, and the ratio of RANKL/OPG. CTX-1 is a marker of bone breakdown that is increased in the blood of postmenopausal women [36,37,38]. BALP is a maker of osteoblast activity elevated in the blood of postmenopausal women [39]. RANKL signalling promotes bone loss, whereas OPG discourages bone loss by binding to and preventing RANKL signalling. The RANKL/OPG ratio is a crucial regulator of osteoclast differentiation, activation, and survival, and thus plays a key role in maintaining the balance between bone formation and bone resorption [40]. CTX-1, BALP, RANKL, and OPG were measured at weeks 0, 12 and 24 in duplicate by enzyme-linked immunosorbent assay (ELISA) according to manufacturers’ instructions: CTX-1 (AC-02F1, Immunodiagnostic Systems), BALP (AC-20F1, Immunodiagnostic Systems), RANKL (RD193004200R, BioVendor R&D), and OPG (EK0480, Boster Biological).

Secondary outcomes

Inflammation and oxidative stress biomarkers

Inflammation and oxidative biomarkers contribute to postmenopausal bone loss and impaired vascular function [8,10,41,42]. High-sensitivity C-reactive protein (hsCRP) is a marker of chronic, low-grade systemic inflammation which is associated with aging, low BMD, and an increased risk of bones fractures. Circulating levels of malondialdehyde (MDA), a byproduct of lipid peroxidation, is a marker of oxidative stress. GSH is the body’s main endogenous antioxidant. Vascular endothelial function declines during menopause, as oestrogens play a crucial role in positively regulating the production of endothelial NO production [10,43] as evidenced by various forms of oestrogen replacement therapy increasing circulating NO levels [44]. The levels of MDA, NO, and GSH were determined spectrophotometrically. hsCRP was measured by ELISA method (CAN-CRP-4360, Diagnostics Biochem Canada). Inflammation and oxidative stress biomarkers were determined at weeks 0,12, and 24.

Statistical analysis

A sample size of 20 participants in each of the six treatment groups was estimated to provide 80% power to detect a clinically significant difference (f = 0.35) in MENQOL group means.

Quantitative measures were summarized as mean ± standard deviation (SD); for baseline values, the range was also provided. Changes in primary outcomes (MENQOL, RI, and BTM) and secondary outcomes (inflammatory markers and ROS) throughout the intervention period were analysed using linear mixed models (LMMs) with random intercepts and slopes, adjusted for age and body mass index (BMI) at baseline. These models included an interaction term between time (in weeks) and randomization group; the coefficients for these interaction terms and their 95% confidence intervals (CI) were reported, representing average differences compared to the placebo group over the entire intervention. Post hoc pairwise contrast analyses were applied to test for significant differences between treatment arms.

BMD T-scores at the end of the intervention were compared between treatment arms using linear regression models, adjusted for baseline T-score, age, and BMI.

The statistical significance threshold was set at 0.05. All analyses were conducted using SAS version 9.4 or R statistical computing version 4.3.2 (R Foundation for Statistical Computing).

RESULTS

A total of 135 subjects were screened, out of which 127 eligible subjects were enrolled into the study (Fig. 1). Four subjects, including two from Ar 250 mg, one from Ar 500 mg, and one from Ws + Ar groups were unable to adhere to the study schedule and dropped out before the first follow-up visit. A total of 123 subjects completed the 24-week dietary supplementation intervention period: placebo (n = 20), Ws 250 mg (n = 20), Ws 500 mg (n = 22), Ar 250 mg (n = 20), Ar 500 mg (n = 20), and Ws + Ar (n = 21). Table 1 presents the mean, SD and range of baseline age and BMI, showing no significant differences between groups.

Fig. 1. Flow chart. Ar: Asparagus racemosus, Ws: Withania somnifera.

Table 1. Baseline characteristics of study subjects.

		Ar 250 mg (n = 20)	Ar 500 mg (n = 20)	Placebo (n = 20)	Ws + Ar (n = 21)	Ws 250 mg (n = 20)	Ws 500 mg (n = 22)	Total (n = 123)	P value
Age (y)									0.865
	Mean ± SD	51.10 ± 3.29	51.30 ± 2.99	50.90 ± 3.13	51.00 ± 3.15	50.10 ± 2.95	50.77 ± 2.64	50.86 ± 2.99
	Range	44.00–56.00	45.00–55.00	45.00–55.00	46.00–55.00	45.00–55.00	45.00–55.00	44.00–56.00
BMI (kg/m2)									0.775
	Mean ± SD	25.78 ± 2.45	26.59 ± 1.87	26.20 ± 2.20	26.55 ± 2.75	26.65 ± 3.13	26.85 ± 2.14	26.44 ± 2.43
	Range	22.60–30.04	24.28–31.76	22.52–30.43	20.88–31.73	22.19–31.26	21.64–30.72	20.88–31.76

Ar: Asparagus racemosus, Ws: Withania somnifera, SD: standard deviation, BMI: body mass index.

Primary outcomes

Table 2 presents the mean ± SD for MENQOL total score and RI primary outcomes at each timepoint, along with the estimated mean difference compared to placebo throughout the trial. A decreasing trend for both outcomes was observed in all treatment groups, showing significant differences from placebo, which remained unchanged during the intervention.

Table 2. MENQOL and RI scores throughout the intervention.

		Baseline	Week 4	Week 8	Week 12	Week 24	Group difference [95% CI]	P value
MENQOL
	Placebo	5.15 ± 0.51	5.15 ± 0.50	5.14 ± 0.51	5.14 ± 0.51	5.11 ± 0.50	-	-
	Ar 250 mg	4.94 ± 0.50	4.88 ± 0.49	4.71 ± 0.47	4.58 ± 0.43	4.28 ± 0.45	–0.03 [–0.03, –0.02]A,C,d,E	< 0.0001
	Ar 500 mg	5.10 ± 0.49	4.89 ± 0.51	4.66 ± 0.51	4.42 ± 0.48	4.04 ± 0.46	–0.04 [–0.05, –0.04]A,B,e,f	< 0.0001
	Ws 250 mg	5.14 ± 0.60	5.02 ± 0.63	4.82 ± 0.64	4.60 ± 0.61	4.24 ± 0.62	–0.04 [–0.04, –0.03]A,b,E	< 0.0001
	Ws 500 mg	5.40 ± 0.71	5.13 ± 0.62	4.92 ± 0.58	4.49 ± 0.55	4.13 ± 0.47	–0.05 [–0.06, –0.04]A,B,c,D,F	< 0.0001
	Ws + Ar	5.20 ± 0.51	5.09 ± 0.52	4.92 ± 0.52	4.73 ± 0.53	4.44 ± 0.56	–0.03 [–0.04, –0.02]A,c,E	< 0.0001
RI
	Placebo	–3.29 ± 0.70	–3.29 ± 0.70	–3.29 ± 0.87	–3.31 ± 0.82	–3.31 ± 0.74	-	-
	Ar 250 mg	–3.21 ± 0.94	–3.74 ± 0.89	–4.27 ± 0.87	–4.93 ± 0.91	–5.66 ± 0.98	–0.10 [–0.12, –0.09]A,C,d,E,F	< 0.0001
	Ar 500 mg	–3.24 ± 0.58	–4.17 ± 0.64	–5.08 ± 0.69	–6.20 ± 0.68	–7.70 ± 0.79	–0.18 [–0.20, –0.17]A,B,D,e,f	< 0.0001
	Ws 250 mg	–3.40 ± 1.05	–3.96 ± 1.09	–4.50 ± 1.12	–5.33 ± 1.17	–6.34 ± 1.17	–0.12 [–0.14, –0.11]A,b,C,E,F	< 0.0001
	Ws 500 mg	–3.37 ± 0.89	–4.60 ± 0.89	–5.70 ± 0.98	–6.84 ± 0.99	–8.45 ± 1.01	–0.21 [–0.22, –0.19]A,B,c,D,F	< 0.0001
	Ws + Ar	–3.29 ± 0.74	–4.07 ± 0.74	–5.02 ± 0.60	–5.84 ± 0.62	–7.17 ± 0.73	–0.16 [–0.18, –0.15]A,B,c,D,E	< 0.0001

Data are presented as mean ± SD and beta coefficients from the LMM arm × time term [95% CI] (weekly group difference).

MENQOL: menopause-specific quality of life, RI: reflection index, CI: confidence interval, Ar: Asparagus racemosus, Ws: Withania somnifera, SD: standard deviation, LMM: linear mixed model, -: not applicable.

Letters indicate significant differences from placebo (a/A), Ar 250 mg (b/B), Ar 500 mg (c/C), Ws 250 mg (d/D), Ws 500 mg (e/E), Ws + Ar (f/F); lower case letters: P < 0.05; capital letters: P < 0.0001.

For MENQOL, score reductions during the intervention period were dose-responsive with higher dosing of each extract contributing to significantly greater reductions, although the combined supplementation of Ws and Ar extracts did not show synergism, as equivalent dosing of each individual extract showed greater reductions in MENQOL than the individual extracts (Table 2 and Fig. 2). The Ws extract performed better than the Ar extract at equivalent dosing in terms of absolute average score reduction. These results suggest daily supplementation Ws or Ar extracts dose-dependently improve quality of life in postmenopausal women.

Fig. 2. Scatter plot and slope of menopause-specific quality of life (MENQOL) score. Ar: Asparagus racemosus, Ws: Withania somnifera.

The analysis of MENQOL domains aligned with the overall MENQOL score results. All treatment groups demonstrated better outcomes than the placebo group across all four domains, although a synergistic effect of Ws and Ar was not observed (Supplementary Table 1, available online). The 500 mg dose of Ws led to a more significant reduction in the psychosocial domain score compared to both the same dose of Ar and the 250 mg dose of Ws. Additionally, a dose-dependent effect was observed for Ws in the physical domain. For Ar, a dose-response effect was noted in the vasomotor, psychosocial, and physical domains. No significant differences were found between groups for the sexual domain.

A similar situation was observed for RI (Table 2 and Fig. 3). All groups supplemented with the Ws and/or Ar extracts showed significant reductions in the RI compared to placebo, indicating reduced vascular endothelial dysfunction with supplementation. The observed improvement in vascular endothelial function during the intervention period was dose-dependent, with higher dosing of each extract contributing to greater reductions in the RI. As for MENQOL, patients allocated to Ws exhibited a greater reduction in RI than the same dosage of Ar; a synergistic effect of both extracts was not observed either in this analysis.

Fig. 3. Scatter plot and slope of reflection index score. Ar: Asparagus racemosus, Ws: Withania somnifera.

Regarding bone metabolism, bone resorption biomarkers CTX, RANKL, and RANKL/OPG BTMs were shown to significantly decreased when compared to placebo, while bone protective OPG plasma values increased across the intervention (Table 3). Again, the greatest reductions were observed in the group supplemented with 500 mg Ws extract. This observation was in accordance with the higher BMD T-score at the lumbar spinal after 24 weeks observed for Ws 500 mg in a lineal model including as covariates age, BMI and baseline T-score, the main predictor of final T-score (Table 4); no significant effect was shown for BMD in the femoral neck measurements (data not shown). Overall, these results suggest dietary supplementation with the Ws or Ar extracts reduce bone turnover and breakdown in postmenopausal women in a dose dependent manner.

Table 3. BTM throughout the intervention.

		Baseline	Week 12	Week 24	Group difference [95% CI]	P value
BALP (ng/mL)
	Placebo	23.86 ± 6.82	23.82 ± 6.82	23.75 ± 6.75	-	-
	Ar 250 mg	20.78 ± 5.33	19.12 ± 5.42	18.43 ± 5.48	–0.09 [–0.17, –0.02]a,C,D,E,f	0.0014
	Ar 500 mg	24.89 ± 7.74	21.42 ± 7.63	17.96 ± 6.71	–0.28 [–0.36, –0.21]A,B,E	< 0.0001
	Ws 250 mg	22.07 ± 4.88	18.01 ± 4.07	13.57 ± 3.55	–0.35 [–0.42, –0.28]A,B,e,f	< 0.0001
	Ws 500 mg	20.99 ± 4.66	15.84 ± 4.05	9.11 ± 1.22	–0.49 [–0.56, –0.42]A,B,C,d,F	< 0.0001
	Ws + Ar	19.78 ± 3.59	16.95 ± 3.42	14.41 ± 3.26	–0.22 [–0.29, –0.15]A,b,d,E	< 0.0001
CTX-1 (ng/mL)
	Placebo	1.84 ± 0.41	1.83 ± 0.40	1.83 ± 0.41	-	-
	Ar 250 mg	1.76 ± 0.30	1.65 ± 0.28	1.49 ± 0.26	–0.01 [–0.02, 0.00]a,c,D,E,f	0.0226
	Ar 500 mg	1.93 ± 0.47	1.60 ± 0.45	1.30 ± 0.55	–0.03 [–0.03, –0.02]A,b,E	< 0.0001
	Ws 250 mg	1.90 ± 0.36	1.52 ± 0.38	1.11 ± 0.25	–0.03 [–0.04, –0.02]A,B,e,f	< 0.0001
	Ws 500 mg	1.95 ± 0.42	1.46 ± 0.29	0.77 ± 0.31	–0.05 [–0.06, –0.04]A,B,C,d,F	< 0.0001
	Ws + Ar	1.86 ± 0.37	1.66 ± 0.33	1.31 ± 0.26	–0.02 [–0.03, –0.01]A,b,d,E	< 0.0001
RANKL (ng/mL)
	Placebo	1,631.40 ± 287.30	1,629.85 ± 288.18	1,630.70 ± 287.55	-	-
	Ar 250 mg	1,755.55 ± 325.59	1,545.05 ± 316.69	1,312.85 ± 258.76	–18.42 [–23.04 , –13.79]A,c,d,E,f	< 0.0001
	Ar 500 mg	1,736.35 ± 306.91	1,403.70 ± 343.28	1,149.30 ± 373.58	–24.43 [–29.05 , –19.81]A,b,E,F	< 0.0001
	Ws 250 mg	1,685.55 ± 261.15	1,454.75 ± 230.50	1,034.85 ± 156.81	–27.08 [–31.71 , –22.46]A,b,E,F	< 0.0001
	Ws 500 mg	1,714.77 ± 291.72	1,222.55 ± 284.37	758.32 ± 234.00	–39.82 [–44.34 , –35.31]A,B,C,D,F	< 0.0001
	Ws + Ar	1,440.62 ± 427.27	1,290.38 ± 406.08	1,192.24 ± 404.11	–10.32 [–14.89 , –5.75]A,b,C,D,E	< 0.0001
OPG (ng/mL)
	Placebo	1,443.39 ± 257.70	1,444.31 ± 251.16	1,448.31 ± 254.22	-	-
	Ar 250 mg	1,670.58 ± 326.99	1,796.95 ± 287.09	1,890.25 ± 302.78	8.95 [4.49, 13.40]a,C,D,E,F	<0.0001
	Ar 500 mg	1,641.25 ± 404.18	2,016.31 ± 421.31	2,304.28 ± 420.22	27.42 [22.97, 31.88]A,B,E,f	<0.0001
	Ws 250 mg	1,571.30 ± 425.80	1,858.67 ± 392.23	2,193.27 ± 342.11	25.71 [21.26, 30.16]A,B,E,f	<0.0001
	Ws 500 mg	1,612.91 ± 397.35	2,082.73 ± 429.04	2,501.71 ± 350.04	36.83 [32.48, 41.18]A,B,C,D,F	< 0.0001
	Ws + Ar	1,605.34 ± 350.46	1,911.90 ± 337.08	2,072.35 ± 316.73	19.25 [14.85, 23.65]A,B,c,d,E	< 0.0001
RANKL/OPG
	Placebo	1.15 ± 0.22	1.15 ± 0.22	1.15 ± 0.22	-	-
	Ar 250 mg	1.10 ± 0.35	0.89 ± 0.27	0.72 ± 0.22	–0.02 [–0.02, –0.01]A,c,D,E	< 0.0001
	Ar 500 mg	1.13 ± 0.37	0.73 ± 0.28	0.52 ± 0.21	–0.02 [–0.03, –0.02]A,b,e,f	< 0.0001
	Ws 250 mg	1.16 ± 0.40	0.82 ± 0.23	0.49 ± 0.12	–0.03 [–0.03, –0.02]A,B,e,F	< 0.0001
	Ws 500 mg	1.12 ± 0.33	0.61 ± 0.18	0.31 ± 0.12	–0.03 [–0.04, –0.03]A,B,c,d,F	< 0.0001
	Ws + Ar	0.93 ± 0.30	0.68 ± 0.20	0.58 ± 0.18	–0.01 [–0.02, –0.01]A,c,D,E	< 0.0001

Data are presented as mean ± SD and beta coefficients from the LMM arm × time term [95% CI] (weekly group difference).

BTM: bone turnover marker, CI: confidence interval, BALP: bone alkaline phosphatase, Ar: Asparagus racemosus, Ws: Withania somnifera, CTX-1: C-terminal telopeptide of type I collagen, RANKL: receptor activator of nuclear factor kappa-B ligand, OPG: osteoprotegerin, SD: standard deviation, LMM: linear mixed model, -: not applicable.

Letters indicate significant differences from placebo (a/A), Ar 250 mg (b/B), Ar 500 mg (c/C), Ws 250 mg (d/D), Ws 500 mg (e/E), Ws + Ar (f/F); lower case letters: P < 0.05; capital letters: P < 0.0001.

Table 4. Lumbar spine BMD T score at the end of the intervention.

	Baseline (week 0)	Final (week 24)	Beta [95% CI]	P value
Placebo	–1.895 ± 0.216	–1.850 ± 0.248	-	-
Ar 250 mg	–1.825 ± 0.343	–1.755 ± 0.382	0.017 [–0.015, 0.050]	0.2951
Ar 500 mg	–1.600 ± 0.346	–1.505 ± 0.382	0.014 [–0.020, 0.048]	0.4144
Ws + Ar	–1.729 ± 0.362	–1.648 ± 0.420	0.015 [–0.017, 0.048]	0.3512
Ws 250 mg	–1.920 ± 0.207	–1.865 ± 0.235	0.012 [–0.021, 0.045]	0.4679
Ws 500 mg	–1.555 ± 0.343	–1.414 ± 0.393	0.054 [0.020, 0.087]	0.0019

Data are presented as mean ± SD and beta coefficients [95% CI] from linear model.

BMD: bone mineral density, CI: confidence interval, Ar: Asparagus racemosus, Ws: Withania somnifera, SD: standard deviation, BMI: body mass index, -: not applicable.

Regression coefficients [95% CI] for covariates: Age –0.0003 [–0.004, 0.003], BMI 0.002 [–0.002, 0.006], Basal T-score 1.120 [1.089, 1.151]. Full model: Adjusted R-squared: 0.981, P value: < 2.2e–16.

Secondary outcomes

The effect of Ws and/or Ar supplementation on inflammatory and oxidative stress markers was also investigated (Table 5). All supplemented groups showed significantly reduced circulating levels of hsCRP, MDA, GSH, and NO, although greater reductions were observed in postmenopausal women supplemented with the Ws extract compared to those supplemented with equivalent dosing of the Ar extract or combination of both.

Table 5. Inflammatory markers throughout the intervention.

		Baseline	Week 12	Week 24	Group difference [95% CI]	P value
hsCRP (mg/L)
	Placebo	2.65 ± 0.33	2.69 ± 0.34	2.69 ± 0.33		-
	Ar 250 mg	2.44 ± 0.41	2.41 ± 0.40	2.39 ± 0.40	0.00 [–0.01, 0.00]a,C,D,E	0.0379
	Ar 500 mg	2.68 ± 0.36	2.56 ± 0.33	2.45 ± 0.34	–0.01 [–0.01, –0.01]A,B,d,E,f	< 0.0001
	Ws 250 mg	2.46 ± 0.43	2.31 ± 0.38	2.15 ± 0.35	–0.01 [–0.02, –0.01]A,B,c,E,F	< 0.0001
	Ws 500 mg	2.62 ± 0.36	2.36 ± 0.30	2.08 ± 0.23	–0.02 [–0.03, –0.02]A,B,C,D,F	< 0.0001
	Ws + Ar	2.52 ± 0.32	2.45 ± 0.30	2.40 ± 0.30	–0.01 [–0.01, 0.00]A,c,D,E	< 0.0001
MDA (µmol/L)
	Placebo	3.74 ± 0.38	3.75 ± 0.36	3.74 ± 0.35		-
	Ar 250 mg	3.45 ± 0.49	3.39 ± 0.47	3.33 ± 0.46	0.00 [–0.01, 0.00]D,E	0.0726
	Ar 500 mg	3.27 ± 0.41	3.18 ± 0.38	3.13 ± 0.36	–0.01 [–0.01, 0.00]a,d,E	0.0311
	Ws 250 mg	3.56 ± 0.48	3.35 ± 0.43	3.17 ± 0.45	–0.02 [–0.02, –0.01]A,B,c,e,f	< 0.0001
	Ws 500 mg	3.30 ± 0.43	2.96 ± 0.27	2.74 ± 0.09	–0.02 [–0.03, –0.02]A,B,C,d,F	< 0.0001
	Ws + Ar	3.38 ± 0.49	3.35 ± 0.49	3.22 ± 0.47	–0.01 [–0.01, 0.00]a,d,E	0.0145
GSH (µmol/L)
	Placebo	575.60 ± 9.49	575.64 ± 9.59	575.71 ± 9.54		-
	Ar 250 mg	574.01 ± 15.15	596.46 ± 16.80	610.16 ± 16.05	1.50 [1.18, 1.83]A,E	< 0.0001
	Ar 500 mg	566.84 ± 12.43	601.51 ± 12.11	610.35 ± 7.58	1.81 [1.48, 2.13]A,E	< 0.0001
	Ws 250 mg	576.28 ± 20.10	606.99 ± 19.46	619.25 ± 18.08	1.79 [1.46, 2.11]A,E	< 0.0001
	Ws 500 mg	578.61 ± 22.95	631.24 ± 9.84	645.39 ± 14.49	2.78 [2.46, 3.09]A,B,C,D,F	< 0.0001
	Ws + Ar	575.61 ± 9.88	597.95 ± 8.96	612.92 ± 9.60	1.55 [1.23, 1.87]A,E	< 0.0001
NO (µmol/L)
	Placebo	31.95 ± 3.00	31.96 ± 3.07	32.02 ± 3.04		-
	Ar 250 mg	34.92 ± 4.03	37.39 ± 4.20	39.00 ± 4.04	0.17 [0.12, 0.22]A,c,E	< 0.0001
	Ar 500 mg	32.57 ± 2.92	36.21 ± 2.83	38.90 ± 3.09	0.26 [0.21, 0.31]A,b,d,e,f	< 0.0001
	Ws 250 mg	34.45 ± 3.81	37.12 ± 4.21	39.31 ± 4.50	0.20 [0.15, 0.25]A,c,E	< 0.0001
	Ws 500 mg	36.40 ± 3.93	41.73 ± 2.69	45.05 ± 2.40	0.36 [0.31, 0.41]A,B,c,D,F	< 0.0001
	Ws + Ar	33.68 ± 3.57	36.63 ± 3.67	38.75 ± 3.74	0.21 [0.16, 0.26]A,c,E	< 0.0001

Data are mean ± SD and beta coefficients from the LMM arm * time term [95% CI] (weekly group difference).

CI: confidence interval, hsCRP: high-sensitivity C-reactive protein, Ar: Asparagus racemosus, Ws: Withania somnifera, MDA: malondialdehyde, GSH: glutathione, NO: nitric oxide, SD: standard deviation, LMM: linear mixed model, -: not applicable.

Letters indicate significant differences from placebo (a/A), Ar 250 mg (b/B), Ar 500 mg (c/C), Ws 250 mg (d/D), Ws 500 mg (e/E), Ws + Ar (f/F); lower case letters: P < 0.05; capital letters: P < 0.0001.

Safety assessment

In terms of safety assessment, one participant in the Ws 500 mg group and three participants in the Ar 500 mg group reported mild gastrointestinal disturbances, which resolved with symptomatic treatment. No participants discontinued the study due to adverse events.

DISCUSSION

Management of menopause and post-reproductive health in women is of increasing importance due to increasing life expectancy. Menopause is a particularly critical time to discuss bone health with healthcare providers due to accelerated bone loss that increases risk for osteoporosis and bone fracture; however, significant awareness and treatment gaps exist [45]. Attention to women with osteopenia is especially warranted to reduce the population burden of since over half of fragility fractures occur in this population [46]. Furthermore, low BMD and elevated BTM are associated with greater fragility fracture risk in postmenopausal women with osteopenia [47]. Therefore, practical therapeutic strategies to address both the psychological and physiological health effects of oestrogen withdrawal, as well as the accompanying increased oxidative stress and inflammation, are essential. Diet is a modifiable lifestyle factor well recognized to influence health, including antioxidant and inflammatory status. Thus, nutrients or nutraceuticals that exhibit antioxidant and anti-inflammatory as well as potential estrogenic activity in the body are expected to attenuate adverse psychological and physiologic health effects that often accompany menopause. We investigated the potential therapeutic effects of two nutraceutical ingredients, standardized aqueous extracts derived from traditionally used therapeutic botanicals in Ayurveda: Ar, primarily recommended for female health, and Ws, commonly used to reduce mental and physical dysfunctions associated with excess stress and anxiety. Both ingredients significantly reduced severity of bothersome menopause symptoms assessed by the MENQOL questionnaire dose-dependently; however, no apparent synergism was observed and overall, the Ws extract led to greater improvements at equivalent dosing. Reductions in systemic oxidative stress and inflammation, vascular dysfunction, bone turnover and loss observed with daily supplementation of the extracts followed this same pattern with improvements being dose-dependent, lacking synergism, and favouring the Ws extract. These results were unexpected, since Ar is an extract traditionally used for its purported phytoestrogenic properties in Ayurvedic medicine and for antioxidant and anti-inflammatory activities [48]. The estrogenic effects of Ar, are attributed to its bioactive compounds, including steroidal saponins and phytoestrogens [22,23,25,41,49]. The studied Ar extract had not previously been evaluated for antioxidant, anti-inflammatory, or phytoestrogenic activity, while other extracts of Ar have been shown to bind to estrogen receptors without modulating endogenous estrogen levels, indicating potential safety in therapeutic use [50]. The effect of Ar on isolated uterine strips from nulliparous nonpregnant rats showed a reduction in contraction force and frequency with more female rats in proestrus suggestive of both anti-oxytocin and (phyto)estrogenic effects [51]. Dyslipidemia and accumulation of cholesterol are more likely during menopause and are associated with estrogen reduction [18]. A herbo-mineral formulation of Ar was found to be hypocholesterolaemic in rats suggestive of cardio-protective activity, although the exact component and mechanism of action of the herbo-mineral formulation involved in this hypolipidemic activity was not identified; indicating further research is needed [52].

Ws has been shown to be cardioprotective by many studies. Histopathology studies of rats with chemicalinduced cardiac injury showed myocardial necrosis, antioxidant enzyme imbalance, and an increase in lipid peroxidation products. However, rats that received Ws had significantly reduced myocardial injury and improved lipid peroxidation and antioxidant enzyme profiles [53]. As Ws is not known to contain phytoestrogens and consists of phytochemicals such as withanolides and flavonoids the proposed therapeutic effects of Ws are based on previously demonstrated antioxidant and anti-inflammatory activity in clinical research [4,22,23,25,41,48]. A constituent of Ws, withaferin A, has demonstrated suppression of estrogen receptor-α protein expression in human breast cancer cells and mediated cell killing via an apoptotic mechanism [54]. However, a recent clinical study in menopausal women taking ashwagandha extract from root showed increased serum estradiol compared to placebo [55]. These opposing studies indicate the need for additional studies with well-defined botanical extracts when standardized to one or more select phytochemical constituents, are compositionally complex and generally contain a vast number of chemical compounds that can be influenced by numerous factors such as growth and harvesting conditions as well as post-harvest processing and extraction methods (e.g., solvents used). Thus, it is unknown which phytochemical constituents within each uniquely complex extract mediated the biological effects Given the profile of both Ar and Ws these extracts pose a good alternative for HRT.

Increased inflammation and oxidative stress associated with oestrogen deficiency play causal roles in vascular dysfunction and bone loss in postmenopausal women [8,10,41,42]. Although hsCRP was significantly reduced by both the extracts, at the higher dose, an ~20% reduction was observed in women supplemented with the Ws extract for 24 weeks vs. an ~9% reduction in those supplemented with the Ar extract. OPG acts as a decoy receptor for RANKL by binding to it and blocking its signalling through the RANK receptor. The ratio of RANKL-to-OPG plays a critical role in the balance between bone formation and breakdown as a key regulator of osteoclast (bone resorbing cells) differentiation, activation, and survival [56]. Inflammation favours an increased RANKL/OPG ratio and bone loss [40]. RANKL was reduced and OPG was increased the most in women supplemented with the higher dose of Ws for 24 weeks, whereas the lower dose of Ws performed similarly to the higher dose of Ar in this regard.

Ovariectomy markedly reduced GSH levels in bone marrow of mice, whereas oestrogen administration restored the depleted GSH levels [42]. Furthermore, administration of N-acetylcysteine (NAC), a GSH precursor, or vitamin C, an antioxidant that supports cellular GSH levels, diminished ovariectomy-induced bone loss, while administration of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, caused depletion of bone marrow GSH levels and bone loss in mice like ovariectomy. In vitro, treatment of osteoclasts with BSO augmented proinflammatory signalling, but treatment with NAC or oestrogen suppressed proinflammatory signalling and the expression of TNF-α [42]. GSH administration to BSO-treated, GSHdepleted osteoblasts significantly reduced the RANKL/OPG ratio via increased OPG mRNA expression [13]. In this study, GSH levels increased the most in women supplemented with the higher dose of Ws for 24 weeks, whereas the lower dose of Ws performed similarly to the higher dose of Ar. Furthermore, MDA levels were reduced ~11% and ~17% after 24 weeks of supplementation with the lower and higher dose of the Ws extract, respectively, compared to only ~3%–4% reductions with Ar extract supplementation. Thus, the Ws extract appeared to exhibit the more potent antioxidant and anti-inflammatory activity in this population, likely underlying the greater reductions observed for vascular dysfunction and bone breakdown in supplemented women.

The percentage change in BTM observed in women supplemented with the lower dose of the Ws extract was also similar or better than observed in women supplemented with the higher dose of the Ar extract, exemplifying the more potent suppression of bone turnover and loss by the Ws extract. Despite changes in BTM slowly translate in changes in BMD, we have shown that patients supplemented with the highest Ws dose had significantly larger T-scores at the lumbar spine at the end of the intervention than patients receiving placebo accounting for baseline T-scores.

Implementing and maintaining significant dietary changes can be challenging, whereas dietary supplementation is generally easier to adopt and sustain. This research establishes two nutraceutical ingredients that have demonstrated efficacy for attenuating bothersome symptoms and adverse health effects of menopause to be considered in addition to other dietary (e.g., calcium/vitamin D supplementation, increase fruit and vegetable consumption) and lifestyle modifications (e.g., physical activity). Diet and physical activity were not monitored during the study, although participants were instructed to maintain habitual diet and activity while dietary supplementation with calcium and vitamin D was not allowed. Thus, intake of dietary components that may influence outcome parameters, such as calcium, is not known, thereby a limitation of this study. Dietary calcium intake by adult Indian women is generally below recommendations [57,58], which can contribute to loss of BMD overtime, yet that was not observed during this 24-week intervention. Another limitation of this study is the relatively small, homogenous sample size. Future research should be considered in larger and different populations of women, such as peri- and post-menopausal women at risk for osteopenia from different geographical regions and racial/ethnic backgrounds, to further evaluate preventative therapeutic potential of these nutraceutical interventions.

Daily supplementation with these unique, aqueous extracts of Ws or Ar support MENQOL, bone mass, and vascular endothelial function in postmenopausal women. These benefits were generally dose-dependent, mediated by attenuation of inflammation and oxidative stress, which contribute to bone loss and vascular endothelial dysfunction in postmenopausal women [8,10,40,41].

SUPPLEMENTARY MATERIAL

Table S1

Menopause Specific Quality Of Life (MENQOL) domains